Calcium Entry Via TRPC6 Mediates Albumin Overload-induced Podocyte Injury

Chronic kidney disease (CKD) is a major public health problem worldwide. Proteinuria is a common clinical signature and a potent predictor for the progression of CKD. Albumin, which is the most abundant component of urine proteins, exerts injurious effects on renal cells in chronic kidney diseases. However, the toxicity of albumin to podocytes is not well elucidated. Podocyte injury is a hallmark of proteinuric kidney diseases. Importantly, the leakage of proteins into the urine is also a primary factor which promotes progressive podocyte injury. Podocyte foot processes and the slit diaphragms are affected when abnormally exposed to a high concentration of albumin in proteinuric conditions, which may worsen podocyte injury, forming a vicious cycle between proteinuria and podocyte injury. The canonical transient receptor potential-6 (TRPC6) channel, which is associated with a subset of familial forms of focal segmental glomerulosclerosis (FSGS) and several acquired proteinuric kidney diseases, was shown to be the most important Ca2+ permeable ion channel in podocytes. However, direct evidence for the causal role of TRPC6-mediated Ca2+ entry in the induction of protein overload-associated podocyte injury is lacking.In the current study, we tested the hypothesis that albumin overload may induce functional and structural changes in podocytes via TRPC6-mediated calcium.Objective To explore the role of TRPC6-mediated calcium entry on albumin-induced podocyte injury. Methods Cultured conditionally immortalized podocytes were divided into several groups:control; vehicle; pEGFP-N1; mTRPC6-EGFP; scramble siRNA; TRPC6 siRNA SKF-96365; Jasplakinolide. (1) Fluo 3-AM (3μM) was used as a calcium indicator and the calcium-dependent fluorescence was monitored using confocal microscope upon albumin stimuli. (2) Western blot analysis was used to detect the protein levels of TRPC6, nephrin, ER stress proteins GRP 78 and cleaved caspase-12. (3) Immunofluorescent staining was used to detect desmin, a marker of podocyte injury. (4) Rhodamine-labeled phalloidin was used to stain the F-actin cytoskeleton. (5) Hoechst 33342 staining was used to detect the changes of nucli in podocytes. (6) Annexin V-FITC and propidium iodide (PI) double staining was used to detect the apoptosis rate.Results high concentration of albumin triggers intracellular calcium ([Ca2+]i) increase through mechanisms involving the intracellular calcium store release and extracellular calcium influx in podocytes. It was found that albumin-induced increase in [Ca2+]i was blocked by TRPC6 siRNA or SKF-96365, a blocker of TRP cation channels. Long-term albumin exposure caused an up-regulation of TRPC6 expression in podocytes, which was inhibited by TRPC6 siRNA. The transfection of TRPC6 siRNA into podocytes or treatment with SKF-96365 also reduced the albumin-induced expression of desmin, which is a marker of podocyte injury. Additionally, the inhibition of TRPC6 prevented the F-actin cytoskeleton disruption that is induced by albumin overload. Moreover, albumin overload induced expression of the endoplasmic reticulum (ER) stress protein GRP78, led to caspase-12 activation and ultimately podocyte apoptosis, all of which were abolished by the knockdown of TRPC6 using siRNA.Conclusion Albumin overload may induce ER stress and the subsequent apoptosis in podocytes via TRPC6-mediated Ca2+ entry.